Effects Of Cold Shock Research Articles

Pore Damage Properties and Permeability Change of Coal Caused by Freeze-Thaw Action of Liquid Nitrogen

A laboratory test was conducted to investigate the effect of the freeze-thaw action of liquid nitrogen on the pore structure and permeability of coal rock. First, coal rock samples with similar sound velocities and permeabilities were selected. These samples were prepared in different water saturation levels and subjected to nuclear magnetic resonance (NMR) test before and after the freeze-thaw action. Furthermore, the freeze-thaw cycle of liquid nitrogen, freezing time, and water saturation of coal rocks were controlled in permeability test. Results showed that the pore diameter, porosity, and permeability of the coal rocks increase after the freeze-thaw action of liquid nitrogen. These characteristics increase further with the increase of water saturation. The fracturing mechanisms of the freeze-thaw action of liquid nitrogen were summarized in two aspects, phase change of pore water and cold shock, and cold shock was mainly discussed. The results indicate that the effect of cold shock is still crucial at low water saturation, but it is limited by the degree of temperature drop. In general, freeze-thaw action of liquid nitrogen can cause damage to pore structure, promote the formation of fracture networks, and consequently improve the permeability of coal rock.

Effects of Cold Shock on Responses of Phosphomonoesters and Free Amino Acids in Phospholipid-Rich Organs in the Amur Sleeper Perccottus Glehni

Similarities and differences were found in the mechanisms of adaptation of the brain and liver to cold shock in the freshwater fish Perccottus glehni. The quantity of phosphoethanolamine in the brain on day 4 of cold shock at +4°C increased 84-fold from the level in controls (+20°C), accounting for 22.3% of the total pool of free amino acids and ninhydrin-positive compounds. Phosphoserine was absent from the brain at +20°C, but cold shock initiated its accumulation to 1.8% of the total pool. The taurine pool at +4°C decreased from 28.3 to 20% of the total pool, yielding first place to phosphoethanolamine. However, in the liver, phosphoethanolamine (PE) and phosphoserine (PS) were not seen in the free form either in controls or at 4°C. The dominant amino acid in the liver at both temperatures was taurine, the quantity at +4°C being significantly increased. Th glutamate pool decreased nine-fold at +4°C. It is suggested that the intense accumulation of PE and PS in the brain at low temperatures is evidence of the specific features of the adaptation of the neuron membrane, reflecting quantitative changes in phospholipids, possibly including sphingosine-1-phosphate.

Effects of cold acclimation on rectal macromorphology, ultrastructure, and cytoskeletal stability in Gryllus pennsylvanicus crickets

Cold-acclimated insects maintain ion and water balance in the cold, potentially by reducing permeability or increasing diffusion distance across ionoregulatory epithelia such as the rectum. We explored whether cold acclimation induces structural modifications that minimize water and ion diffusion across the rectum and maintain rectal cell integrity. We investigated rectal structure and cytoskeletal stability in chill-susceptible adult Gryllus pennsylvanicus crickets acclimated for one week to either warm (25 °C) or cold (12 °C) conditions. After acclimation, we used light and transmission electron microscopy to examine rectal macromorphology and rectal pad paracellular ultrastructure. We also used fluorescence microscopy and a filamentous-actin (F-actin) specific phalloidin stain to compare the polymerization state of the actin cytoskeleton for each of the acclimation groups before and after a cold shock (1 h at −4 °C). Cold acclimation did not alter rectal pad cell density, or the thickness of the rectal pads, muscle, or cuticle. The tortuosity and width of the rectal pad paracellular channels also did not differ between warm- and cold-acclimated crickets. Rectal pad cells had clear basal and apical regions with differing densities of F-actin. Cold shock reduced the density of F-actin in warm-acclimated crickets, whereas cold-acclimated crickets appeared to have unchanged (basal) or enhanced (apical) F-actin density after cold shock. This suggests that while cold acclimation does not modify rectal permeability through structural modifications to increase diffusion distance for water and ions, cold-acclimated crickets have a modified cytoskeleton that resists the depolymerising effects of cold shock.

Giving eyespots a shiner: Pharmacologic manipulation of the Io moth wing pattern

Our knowledge of wing pattern formation in Lepidoptera has advanced significantly in recent years due to the careful examination of several groups of butterflies. The eyespot is a prominent feature of Lepidoptera wing pattern, especially in the family Saturniidae. The present study examined how sulfated polysaccharides, which are known to simulate cold shock effect in nymphalid butterflies, affected the wing pattern formation of the Io moth, Automeris io (Saturniidae). Prepupae and pupae of this species were subjected to injections of heparin and cold shock. While the cold shock had little to no effect on wing pattern, the aberrations resulting from heparin injections consisted of moderate to profound increases in melanism around the eyespots. The resulting aberrations are dubbed 'Black Eye' and 'Comet Eye.' Most other known aberrations of Automeris io eyespots are summarized, illustrated and named.

Effect of cold and heat shock treatments on the response ofBrucella melitensisto various environmental stresses

The effect of cold shock (10 °C for 2 h) and heat shock (42 °C for 45 min) treatments on the viability of Brucella melitensis under adverse conditions was investigated. Viability of B. melitensis cells during storage at 4 °C was significantly affected by cold shock and heat shock treatments. While cold-shocked cells became more resistant to cold storage, a significant decrease in the viable population of heat-shocked cells was observed. Similarly, cold-shocked cells of B. melitensis became more resistant and heat-shocked cells became more susceptible to acidic conditions. Although cold shock treatment did not affect the thermal tolerance of B. melitensis, heat shock treatment increased significantly the thermal tolerance of the bacterium. Nevertheless, these shock treatments, under the conditions tested, did not alter the susceptibility of B. melitensis to incubation at −18 °C and the presence of 10% NaCl. Practical applications In the food industry, cooling chains and heating processes could potentially induce a cold or heat shock phenomenon. Hence, this information should be taken into account in the development of food preservation measures and for conducting risk assessments on the importance of this pathogen in food processing operations.

A 31-kDa seminal plasma heparin–binding protein reduces cold shock stress during cryopreservation of cross-bred cattle bull semen

In the present study, a 31-kDa protein, purified from cattle bull seminal plasma heparin–binding proteins (SP-HBP), was characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and mass spectrometry. Raw semen of six cross-bred bulls was treated with 31-kDa HBP before cryopreservation to observe its effect on motility, viability, hypo-osmotic swelling test, acrosome integrity, in vitro capacitation/acrosome reaction, and oxidative stress at pre-freeze and frozen–thawed phases of cryopreservation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of 31-kDa protein eluted and purified from SP-HBP (separated on acrylamide gels) resulted in a single band of 40 kDa. In matrix-assisted laser desorption/ionization–time of flight analysis, 12 peptides were identified with matching significantly (P < 0.05) to interlukin-6 of bovine with a top score of 55. Addition of 25 μg/mL of fluorescein isothiocyanate–conjugated 31-kDa protein to raw semen and incubation at 37 °C for 20 minutes before cryopreservation resulted in its binding mainly to head region. Treatment of semen with 31-kDa HBP resulted in a significant (P < 0.05) average increase of 9.2%, 6.8%, and 11.7% and 5.5%, 6.5%, and 11.0% in motile, viable, hypo-osmotic swelling–responsive spermatozoa in six bulls at pre-freeze and frozen–thawed phases of cryopreservation, respectively. Percentage of spermatozoa with intact acrosomes nonsignificantly enhanced in the semen treated with 31-kDa HBP at both phases of cryopreservation. An average nonsignificant increase of 3.1% in in vitro capacitated and acrosome-reacted spermatozoa was obtained in semen supplemented with 31-kDa HBP. Addition of 31-kDa HBP also nonsignificantly reduced Malonadialdehyde (MDA) level by 10.7 and 19.3 μM/109 spermatozoa in prefrozen and frozen–thawed semen, respectively. The results obtained here indicate to conclude that treatment of cross-bred cattle bull semen with 31-kDa HBP protects the spermatozoa from cold shock effect by coating the sperm surface.

Effects of cold-shock triploidy induction treatment on cell cleavage, hatching rate, and survival of triploid larvae of spotted sand bass (Paralabrax maculatofasciatus)

Three experiments assessed the effects of cold-shock induced triploidy on (1) the incidence of abnormal cell cleavage during embryogenesis, (2) hatching success and the proportion of live yolk-sac larvae in normally cleaved versus abnormally cleaved embryos, and (3) survival of triploid larvae of spotted sand bass (Paralabrax maculatofasciatus). All spawns were obtained by hormonal induction (LHRHa). Triploidy was induced by cold shock shortly after fertilization. In experiment 1, the percentage of abnormal cell cleavage was significantly higher (P &lt; 0.05) in the cold-shocked group than in the control group, while the hatching rate and the proportion of live yolk-sac larvae were significantly lower (P &lt; 0.05). In experiment 2, when normally and abnormally cleaved embryos from the control and cold-shocked groups were incubated separately, normal cleaved embryos in the cold-shocked group showed a significantly higher hatching rate and proportion of live yolk-sac larvae than abnormally cleaved embryos in the same group. No difference was observed in the control group. In experiment 3, a significant decrease (P &lt; 0.05) in the triploidy count was observed throughout the larval period. Final survival was drastically lower for triploid larvae compared to cold-shocked diploids and untreated eggs. Low triploidy percentages at the end of the larval period would seriously hinder the development of triploid sand bass culture.

Changes in the fatty acid composition and regulation of antioxidant enzymes and physiology of juvenile genetically improved farmed tilapia Oreochromis niloticus (L.), subjected to short-term low temperature stress

We evaluated the effect of cold shock on the fatty acid composition, antioxidant enzymes, and physiological responses of genetically improved farmed tilapia (GIFT). Experimental GIFT tilapia, a warm-water teleost, were initially acclimated at 28°C and then transferred directly to 13°C. Stress responses were monitored for 120h. There was a significant change in all parameters in response to the cold stressor (P<0.05). Serum cortisol levels increased from 336.93ng/ml to a peak of 1165.31ng/ml 24h after the initial cold shock, and declined rapidly thereafter. Serum glucose and cholesterol levels were significantly lower in the low temperature group than the control group at 120h (P<0.05). Acute low temperature stress enhanced superoxide dismutase, glutathione peroxidase, catalase, and glutathione levels in the liver of GIFT tilapia. The GIFT tilapia were able to selectively metabolize fatty acids for energy needs during the early period of exposure to low-temperature stress. During this time, they primarily used saturated fatty acids for energy. However as the duration of the stressor and loss of muscle fat increased, the fish began to metabolize long-chain polyunsaturated fatty acids. Increased malondialdehyde was produced by oxidation of these fatty acids leading to oxidative damage. Our results provide insight into the changes in fatty acid metabolism physiology that allow GIFT tilapia juveniles to adapt to short-term cold stress.

Effect of cold shock and hormone on growth and male morphotypes of freshwater prawn,Macrobrachium rosenbergii(de Man)

A study was carried out to determine the effects of cold shock and 17α-methyl testosterone hormone on growth in male morphotypes of giant freshwater prawn Macrobrachium rosenbergii. Juvenile males were exposed at 18 ± 0.25°C for 24 h with normal feed and cultured at 27 ± 0.7°C for 70 days as well as administrated feed with hormone separately at 400 mg kg−1 feed for the first 30 days of culture period (70 days). The growth (Mean ± SD) of male juveniles at harvest was closely homogenous in the cold shock (CLS) treated group; while it was heterogeneous in hormone-treated (MH) and control (CO) groups. Although total body weight of blue claw (BC) males was not significantly different in the controls and the treatments, the 2nd pereiopods (claw) weight and length of the BC was significantly lower in CLS (0.52 ± 0.05 g, 10.02 ± 0.65 cm) than that in the CO (0.71 ± 0.16 g, 12.53 ± 0.57 cm) and MH (0.75 ± 0.08 g, 12.85 ± 1.18 cm) treatments. Survival of prawns was similar in treatments. The morphotypes of male prawn were significantly different with 59% of BC males with small-sized claws in CLS and, 45% and 30% of BC males with large-sized claws in MH and CO respectively. Cold shock adversely affected male morphotypes of the prawn so that numbers of BC males with small-sized claw was the highest in CLS tank. However, the CLS treatment resulted in more uniform marketable prawns and suggested a high production of BC males in an all-male culture system.

Pressure resistance of cold-shocked Escherichia coli O157:H7 in ground beef, beef gravy and peptone water.

(i) To study the effects of cold shock on Escherichia coli O157:H7 cells. (ii) To determine if cold-shocked E.coli O157:H7 cells at stationary and exponential phases are more pressure-resistant than their non-cold-shocked counterparts. (iii) To investigate the baro-protective role of growth media (0·1% peptone water, beef gravy and ground beef). Quantitative estimates of lethality and sublethal injury were made using the differential plating method. There were no significant differences (P>0·05) in the number of cells killed; cold-shocked or non-cold-shocked. Cells grown in ground beef (stationary and exponential phases) experienced lowest death compared with peptone water and beef gravy. Cold-shock treatment increased the sublethal injury to cells cultured in peptone water (stationary and exponential phases) and ground beef (exponential phase), but decreased the sublethal injury to cells in beef gravy (stationary phase). Cold shock did not confer greater resistance to stationary or exponential phase cells pressurized in peptone water, beef gravy or ground beef. Ground beef had the greatest baro-protective effect. Real food systems should be used in establishing food safety parameters for high-pressure treatments; micro-organisms are less resistant in model food systems, the use of which may underestimate the organisms' resistance.

Preservation of spotted buffalo epididymal spermatozoa in andromed with raffinose addition

Raffinose is an external cryoprotectant that would protect cell membrane from cold shock effect during preservation process. This study was perform to observe the effect of raffinose on the quality of spotted buffalo epididymal spermatozoa during storage at 4 °C for 24 h. Three different extenders, i.e. commercial tris-egg yolk free extender (Andromed) as control (AM), Andromed Andromed plus 0.2% w/v raffinose (AMR0.2), and Andromed plus 0.4% w/v raffinose (AMR0.4), were used in this study. The results showed that the progressive motility and viability of spermatozoa reduced significantly during storage period. At 12 h of storage, the progressive motility was 60.33% in AM versus 60% in both AMR0.2 and AMR0.4 (P>0.05). Meanwhile, after 24 h of storage, the progressive motility in both AM and AMR0.4 extenders was 55% versus 55.33% in AMR0.2 (P>0.05). Spermatozoa viability in AM, AMR0.2, and AMR0.4 were 70.30%, 73%, 72.33% after 12 h of storage and 66.33%, 68.67% and 69.67% after 24 h of storage, respectively (P>0.05). This results suggested that Andromed without an external cryoprotectant addition could maintain the quality of epididymal spermatozoa as good as the one with 0.2% and 0.4% raffinose addition.

Water-deficit tolerance induction during germination of Jalo Precoce bean (Phaseolus vulgaris L.) cultivar

Bean (Phaseolus vulgaris L.) cultivars are susceptible to water stress, especially during germination. Salicylic acid and thermal shock improve tolerance level in this plant to different kinds of stresses. However, in the present work, effects of cold shock and salicylic acid alone or combined on germinating beans under imposed water deficit were evaluated. Bean seeds from the Jalo Precoce cultivar were imbibed either in water or in salicylic acid for 24 h. After treatment, lots were divided and exposed to cold shock (7 °C) for another 24 h. The seeds were then placed on paper rolls imbibed with different concentrations of mannitol to impose a water deficit on them. They were evaluated for germination; seedling vigour; dry and total weights and length of shoot root; superoxide dismutase activity and proline content. Proline accumulates as a response to water-deficit stress, and SA alone or combined with cold shock improves this response. The use of SA increases many of the physiological variables than cold shock and decreases the antioxidant activity of SOD.

Physiological and behavioural consequences of cold shock on bonefish (Albula vulpes) in The Bahamas

The effects of cold shock on fish have rarely been assessed in sub-tropical regions despite the fact that such events can occur as a result of upwelling, storms, or other climactic events that are expected to increase as a result of global environmental change. Here, the sub-lethal physiological and behavioural consequences of cold shock on bonefish (Albula vulpes) were assessed using four treatment groups (control, handled control, 7°C below ambient, and 14°C below ambient conditions; n=8 fish/treatment). The control and handled control remained at ambient temperature (~24°C) while other treatments were exposed to a 2h cold shock. Handled controls were treated with the same handling procedures as cold shock treatments, while control fish were less disturbed. Ventilation rate and reflex indicators (e.g., equilibrium, response to stimuli) were recorded throughout the experiment, and swimming ability was evaluated 2h after cold shock exposure. A blood sample was taken after the cold shock exposure and 2h following the swimming assessment to quantify glucose and lactate concentrations, hematocrit and pH in whole blood, and cortisol titers from spun plasma. Bonefish exposed to 14°C below ambient temperature experienced elevated lactate concentrations and high reflex impairment, with the majority of fish losing equilibrium <30min into exposure. During cold shock exposure, ventilation rates were significantly lower in the 7°C below ambient treatment than in the handled control. After cold shock exposure, swimming ability decreased as the magnitude of cold shock increased, however, it was also depressed in the handled control relative to unhandled controls. Exposure to 14°C below ambient temperatures had significant physiological and behavioural impacts on bonefish, while 7°C below ambient had little effect. Abrupt declines in water temperature in shallow tropical or sub-tropical seas >7°C due to storm events or upwelling have the potential to cause physiological and behavioural impairment that could lead to mortality in this species.

Evaluation of cold shock-induced cytotoxicity and genotoxicity in the house fly Musca domestica

Background: Low temperature affects the survival, growth and development of invertebrates, especially insects, based on the severity of cold and the duration of exposure. Although the effects of cold shock or direct chilling were previously analysed in terms of development patterns and defects, morphological changes, cold hardiness, cryopreservation and diapause in insects, very little information is available regarding the effects of cold shock at the chromosomal level. Material and Methods: Late third instar larvae of the house fly Musca domestica were exposed to low temperatures (10, 4, 0 and -5°C) for different durations, in order to assess genotoxicity and cytotoxicity in the present study. The chromosomal aberration assay and micronucleus test were used as genotoxic end points. Cytotoxicity was evaluated by the mitotic index and the extent of tissue damage was observed using the Trypan blue staining method. Results: A significant (P Conclusions: The present work suggests that cold shock induces chromosome aberrations and cytotoxicity and affects the developmental pattern in house fly, M. domestica.

Effects of cold shock on host egg parasitization by females of Trichogramma buesi Voegele (Hymenoptera, Trichogrammatidae)

The effects of cold shock (4°C for 18–24 h) on survival of Trichogramma buesi females, their fecundity, and the inclination to parasitize grain moth eggs were studied under laboratory conditions. Cold shock did not result in any significant change in the survival rate, whereas the fraction of females that parasitized grain moth eggs and the fecundity of these females slightly decreased. However, females which had already started oviposition before cold shock infested grain moth eggs much more frequently. In most insect species studied, cold shock results in a sharp decrease or even disruption of the effect of experience on the subsequent behavior. Thus, the results of this study suggest that “the effect of acquired experience” (a tendency to continue infestation of the particular host species) in Trichogramma females is based not on learning or not only on learning but on some other, possibly hormonal mechanisms.

Cold-shock-induced polyspermy and low-temperature tolerance in Japanese sea urchin eggs

Low temperature of seawater is critical for success of fertilization and reproduction in some species of sea urchin. Cortical reaction was prevented and polyspermy was induced in sea urchin eggs subjected to the cold-shock in selective species. By the observation with two fluorescent probes connected phalloidin and lectin, it was revealed that the cortical granules moved to deeper layer from surface in the eggs after the cold-shock treatment, and the signal of actin filaments at the surface were detected more sharply than those in the control eggs. The degree of effects of cold-shock varied among the species of sea urchin. High sensitivities to cold-shock were observed in some sea urchins with reproductive seasons in summer. In contrast, several sea urchins from the northern part of Japan or those species with reproductive seasons in winter were insensitive to the cold-shock. A stronger tolerance to low temperature was observed in the eggs of the family Strongylocentrotidae by the phylogenetic analysis of Japanese sea urchins. This tolerance to low temperature may be influenced on evolutional processes. Recently, it has been reported that a part of this family Hemicentrotus pulcherrimus are distributed in the more northern areas in the Sea of Japan. Tolerance to low temperature may confer advantages to certain species of sea urchins in terms of distribution and reproductive season.

The Production of Triploid Clariobranchus in Indoor Hatchery

A study was carried out to determine the effect of cold shock on hybrid Clariobranchus on the process of producing triploid. One Heterobranchus bidorsalis male broodstock of weight 2kg and age 36 months was selected to cross with one Clarias gariepinus female broodstock of weight 1.5kg and age 12- 15months. The female was injected above the lateral line with hormone (ovaprim) at rate of 0.5ml/kg after a latency period of 12 hours.Fish with eggs freely oozing out on slight touch were stripped into a dry bowl, the eggs were fertilized and divided into two parts in duplicates (A1, A2, B1, B2) and incubated. Triploidy was induced by subjecting the fertilized female eggs to a temperature of 5 o c for 3(three) to 4(four) minutes. A total of 100 fries was stock in each water rearing receptacle and initial weight of diploid is (0.0019±0.001) stocked in B1 and B2 and triploid treatments (0.0019±0.001) stocked in A1 and A2, fries was counted to monitor their daily survival and growth rate on weekly basis for eight weeks. The final weights of diploid were (0.756±0.161) and triploid (1.62±0.080) and their percentage of survival rate were 48% for triploid and 70% for diploid. The change in length for triploid (3n) was from 0.68cm to 5.12cm and the diploid fish from 0.54cm to 5.00cm.The growth rate was monitored and analysis of variance (ANOVA) was used to record the change in weight and length of fish. The triploid strain performs better in growth than diploid strain.

The susceptibility of Streptococcus thermophilus 14085 to organic acid, simulated gastric juice, bile salt and disinfectant as influenced by cold shock treatment

Streptococcus thermophilus is a thermophilic lactic acid bacterium which is used as the starter organism for the fermentation of yoghurt and some cheese. In the present study, S. thermophilus BCRC 14085 was subjected to cold shock treatment by exposure at 10 °C for 2 h. The effect of cold shock on the susceptibility of S. thermophilus in subsequent lethal stress environments such as simulated gastric juice (pH 2.0–3.0), bile solution (2.0%) and various organic acids (0.75 M, pH 3.5) including propionic, lactic, acetic, citric and tartaric acid was investigated. In addition, the survival of cold-shocked and non-shocked S. thermophilus exposed to disinfectants, Clidox-S and Quatricide, were compared. Results revealed that cold shock enhanced the tolerance of S. thermophilus in the presence of simulated gastric juice (pH 2.5 and 2.8), while in bile solution, the population increase of cold-shocked cells is higher than that of non-shocked cells after 12 h of incubation. Furthermore, the susceptibility of S. thermophilus, regardless of cold shock, to organic acid varied with the kinds of organic acid examined. The cold-shocked S. thermophilus showed a significantly less survival (P < 0.05) than that of the non-shocked cells when exposed to lactic or acetic acid. Furthermore, cold shock reduced the survival of S. thermophilus when exposed to Quatricide but not Clidox-S.

Effect of a cold shock on the activity and composition of the communities of ammonium-oxidizing microorganisms in a chestnut soil

The simulation of a cold shock was performed in an incubation experiment with soil microcosms by a sharp decrease of the temperature to negative values and the subsequent analysis of the nitrification rate of the ammonium-oxidizing microorganisms. Three procedures of the cold shock effect were selected: long, short-time, and cyclic. A significant decrease of the nitrifying activity was recorded after the long effect, whereas the 8-, 16-, and 24-hour cold shocks did not affect the intensity of nitrification. A cyclic temperature decrease alternating with periods of incubation under high temperatures also did not affect the nitrifying activity of the microorganisms. We suppose that the domination of mesophilic microorganisms with a resistant enzyme system or of psychrophilic and psychrotolerant microorganisms contributes to the preservation of a high nitrification level in soils with frequent alternations of high and low temperatures.

The effect of cold shock on the free amino acid pool of rotan pondfish Perccottus glehni (Eleotridae, Perciformes)

The effect of cold shock on the free amino acid pool of the pondfish P. glehni not adapted to hibernation has been studied. In the beginning of September, after four days of cold shock, a decrease in the blood level of essential amino acids and an increase in the level of alanine and three-fold increase in the level of γ-aminobutyric acid (GABA) were observed. Changes in the free amino acid pool in the mussels occur in two stages. Namely, in a considerable decrease in their concentration after two days of cold shock followed by an even more significant increase in their amount after four days of shock. In the brain changes in the free amino acid pools are associated with their total decrease followed by continuous significant decrease in the level of taurine during the first four days of cold shocking. The obtained results are compared with previously obtained data on the free amino acid content in muscles and blood of P. glehni during preparation for winter sleep and the beginning of hibernation.